KDM2B regulates hippocampal morphogenesis by transcriptionally silencing Wnt signaling in neural progenitors

The hippocampus plays major roles in learning and memory, and its formation requires precise coordination of patterning, cell proliferation, differentiation, and migration. Here we removed the chromatin-association capability of KDM2B in the progenitors of developing dorsal telencephalon (Kdm2b∆CxxC) to discover that Kdm2b∆CxxC hippocampus, particularly the dentate gyrus, became drastically smaller with disorganized cellular components and structure. Kdm2b∆CxxC mice display prominent defects in spatial memory, motor learning and fear conditioning, resembling patients with KDM2B mutations. The migration and differentiation of neural progenitor cells is greatly impeded in the developing Kdm2b∆CxxC hippocampus. Mechanism studies reveal that Wnt signaling genes in developing Kdm2b∆CxxC hippocampi are de-repressed due to reduced enrichment of repressive histone marks by polycomb repressive complexes. Activating the Wnt signaling disturbs hippocampal neurogenesis, recapitulating the effect of KDM2B loss. Together, we unveil a previously unappreciated gene repressive program mediated by KDM2B that controls progressive fate specifications and cell migration, hence morphogenesis of the hippocampus.

Regarding the analysis of the DG phenotype, the authors mention that there are fewer neurons and stem cells in mutants, however it is unclear whether this relates to the DG being smaller (and this reduction is not quantified either and should be) or if in the smaller DG these cell types are less represented than in the control. Moreover, numbers of HOPX+, PROX1, and DCX+ cells are not shown.
What is the expression pattern of KDM2B during DG development? Which cell type express it and when? This could help defining its role better.
There appears to be an ectopic formation of a scaffold in the DNE region, along the secondary matrix ( Fig.3A-B) which could explain why more progenitors are generated and can delaminate and migrate but are unable to reach their destination, unsupported as this abnormal scaffold does not expand to the forming DG. The author did not quantify or examine these supporting GFAP cells (difficult to see on the composite panels) which normally exclusively form in the fimbria. It would be interesting to investigate, or at the very least discuss, this aspect further.
Regarding the CA phenotype, a reduction of calbindin positive cells is shown in Fig.1. Later, in Fig.6 the effect of Wnt genes and Sfrp2 overexpression are examined by IUE in this domain. The CA phenotype could be further explored while, in contrast, effect of the electroporation should have been investigated in the DG region.
Regarding the behavioural characterisation of the cKO animals, the moving tracks from the open field test (Fig. 2M) show very different patterns in the cKO and significant reduction in their distance and mobility time, while the quantification shows no differences (Fig. S3.A), please explain this apparent discrepancy.
While reporter analyses indicate that Nestin-CreERT2 (Fig. S3) is active on the reporter allele, this does not necessarily mean that deletion of the Kdm2b exon was as efficiently induced. Striking differences in deletion efficiency exist between different alleles, and this is particularly true for inducible Cre which are only active during tamoxifen treatment. Therefore, unless deletion of the targeted allele is shown, these experiments are suggestive but not conclusive.
Regarding the molecular analysis of cKO DG development It is unclear from the text why the genes in Fig. 6.D-G were picked for analysis. Probably because these are Wnt pathways genes mostly enriched in CpG islands? If so, authors should mention it in the text. From the ATAC profiles in Fig. 6.H and S8, chromatin do not seem particularly more open in the loci shown. Authors should comment on this, and improve representations, the figures are quite difficult to read (lots of data but very difficult to see differences between mutants and controls). Is Lef-1 profile exclusively affected in neurospheres (Fig.S8K)?
Although not pivotal for the main scope of the paper, data shown in Fig. S9.A-G is not particularly straightforward. The line of the side of panel A ("High in Ctx") appears too short and should go up to Wnt6. The interpretation provided, regarding the differential effect of Wnt signalling dysregulation by KDM2B in the cortex vs. the hippocampus, is not supported by the data presented. Many Wnt genes are indeed clearly upregulated in the cortex. Are there statistically more Wnt pathway DEGs in DG compared to the cortex?
Regarding the generation of Rnf2 mouse line for analysis of Ring1B role in DG development, comes a bit unexpected, while being useful for exploring the specific role of KDM2B in DG development. Authors should mention Ring1B and its function in the intro already to facilitate the inclusion of this chapter. In addition, histone modifications shown in Fig. S9.I should be quantified.
Finally, given the unexpected results from these Rnf2 mutants, the potential role of KDM2B for DG development should be better discussed. The graphical abstract suggests a potential role for PRC1 while the Rnf2 mutant phenotype may suggest otherwise?
Minor comments: -Line 68: define CBX proteins. -Line 68,69: should put "canonical PRC1" and "variant PRC1" in brackets to improve readability. - Fig. 5 should correct to "upregulated" and "biological process". -Line 134-141: S1C is referred but should be replaced by S2C. -Line 150: the authors indicate data not shown, but it seems to refer to Fig.3E? -Cartoons used in Fig.3.B,D,J; 4.B; S4.B,D; S5.B; S9.J are strikingly similar to Caramello et al., 2021 and should therefore be referred to as "adapted from". -In line 280, the length of the EdU pulse for the analysis at E16.5 should be mentioned. -In Fig. S6 the title should be modified as "abnormal/precocious neuronal differentiation is not responsible…. " -"a tad", "climax", "block of their attenuation" should be replaced.
-Please define all abbreviations Reviewer #2 (Remarks to the Author): In this manuscript, Zhang, et al have discovered that KDM2B plays a crucial role in regulating hippocampal morphogenesis by silencing Wnt signaling in neural progenitors. When the chromatinassociation capability of KDM2B was removed in developing dorsal telencephalon, the hippocampus became drastically smaller with disorganized cellular components and structure. KDM2B mutations in mice resulted in defects in spatial memory, motor learning, and fear conditioning. The migration and differentiation of neural progenitor cells were greatly impeded in the developing hippocampus. The study suggests that KDM2B is essential for proper hippocampal formation and function. The manuscript presents several concrete advances in understanding the role of KDM2B in hippocampal morphogenesis. Overall, I think this is valuable manuscript.
1. The behavioral tests used in the study, such as the Morris water maze and fear conditioning, provide valuable insights into the functional consequences of KDM2B mutations. However, these tests have their own limitations and may not fully capture the complexity of behaviors or cognitive functions that could be affected by KDM2B mutations.
2. The study found that removal of KDM2B's chromatin association capability in adult neural stem cells exerts no effect on adult neurogenesis of the dentate gyrus. This could be seen as a limitation as it suggests that the role of KDM2B in neurogenesis may be limited to the developmental stage.
Reviewer #3 (Remarks to the Author): In the manuscript by Zhang et al. authors evaluated the development of the hippocampus when removing the chromatin association capability of KDM2B (Kdm2bdeltaCxxC, henceforth deltaCxxC) in progenitor cells of the developing dorsal telencephalon. They found an abnormal morphology of the hippocampus in adult deltaCxxC conditional KO (cKO) mice, particularly in the dentate gyrus which was smaller. deltaCxxC mice also showed cognitive an motor impairments. The proposed mechanism involves the increase in the expression of several components of the Wnt signaling pathway that were de-repressed. These are very interesting and novel findings. Most of the conclusions are well supported by the results obtained.
Major comments: 1. A description of the expression patterns of Emx1 and Nestin must be included, as well as a discussion of the differences observed in the survival of both conditional KO (cKO) mice. What is the phenotype of Nestin-deltaCxxC (compared to Emx1-deltaCxxC) at P0 and P7? 2. The same analysis shown in Fig. S1H should be carried out in Emx1-deltaCxxC 3. Authors determined that a strong decrease in GFAP+SOX2+ NSCs, HopX+ NSCs, TBR2+ and DCX+ (which should be quantified as done for NeuN+ and GFAP+SOX2+) cells in the dentate gyrus of adult deltaCxxC animals. However, they conclude that deletion of CxxC ZF of KDM2B had no effect on adult neurogenesis of the DG based on the conditional deletion in adult animals. Authors should improve the discussion of these results. 4. The density (per GCL area or volume) of GFAP+SOX2+ NSCs, HopX+ NSCs, TBR2+ and DCX+ cells should be quantified to demonstrate whether the decrease of these cells is due to the shorter DG.
5. Authors conclude that deltaCxxC mice exhibit hippocampal agenesis. I suggest changing "agenesis" since although smaller, the hippocampus is present in cKO mice.
6. Figure 2F is not mentioned in the Results section or discussed. What can be concluded of this analysis where no differences in the number of platform crossing were observed in cKO mice. The representative swim path of cKO mice during the probe trial ( Fig 2C) should be change; it does not seem representative based on the graph. 7. Line 235-237: "Together, loss of KDM2B-CxxC impedes migration and differentiation of IPCs, hence proper production and localization of granule neurons during hippocampal formation." How did authors conclude with the results of Figure 3 that there was an impairment in differentiation. The effects in PROX1+ cells could be caused only by the altered migration; and is it possible to exclude a proliferation impairment? Total number of EdU+ cells and the percentage of EdU+ cells positive for PROX1 should be evaluated.
8. Line 288-289: "In summary, the migratory and differentiating trajectory of hippocampal progenitors were greatly delayed upon loss of KDM2B-CxxC". What do authors mean by delayed differentiating trajectory? Considering that as shown in Fig 4 "the differentiating rate from RGCs to IPCs (PAX6+TBR2+/PAX6+) at FDJ is 60% higher in cKOs". 9. Line 298-301: "Together, KDM2B regulates hippocampal morphogenesis by controlling multiple behaviors, including coordinated RGC to IPC differentiation, migration, and divisions of neural progenitors (Fig. S6C)." What is the evidence to conclude that KDM2B regulates divisions of neural progenitors? The discussion of the evidence supporting the specific effects (or roles of KDM2B) on migration, differentiation, proliferation should be improved. -Fig3I, y-axis "Total" not "Totol" -A brief description of the markers used should be given in the results section when describing the IFs (e.g. ZBTB20).

Reviewer #1 (Remarks to the Author):
In this study entitled "KDM2B regulates hippocampal morphogenesis by We thank the reviewer to point this out and performed additional staining and measurements. In revision, we measured the length and area of DGs to find Kdm2b cKO hippocampi had significant smaller DGs ( Supplementary Fig.2d-s2g). The decrease of numbers of NeuN+ neurons in cKO animal was comparable to the decrease of DG length or area. Interestingly, the cell densities of HopX+ and SOX2+GFAP+ stem cells were significantly decreased upon deletion of KDM2B-CxxC ( Supplementary Fig.2m-2n).
3. What is the expression pattern of KDM2B during DG development? Which cell type express it and when? This could help defining its role better. This is also a very important issue. We first performed qRT-PCR of Kdm2b using developing hippocampal tissues. The expression of Kdm2b showed an expression plateau between E16 and P0, when hippocampal morphogenesis and neurogenesis are at the peak ( Supplementary Fig.1i). Then Kdm2b ISH were performed onto P0 hippocampal sections followed by TBR2 immunofluorescent staining. Data showed many Kdm2b-expressing DG cells also express TBR2, strongly suggesting Kdm2b's role in these cells ( Supplementary Fig.1j). Related texts were modified or added accordingly.
4. There appears to be an ectopic formation of a scaffold in the DNE region, along the secondary matrix ( Fig.3A-B) which could explain why more progenitors are generated and can delaminate and migrate but are unable to reach their destination, unsupported as this abnormal scaffold does not expand to the forming DG. The author did not quantify or examine these supporting GFAP cells (difficult to see on the composite panels) which normally exclusively form in the fimbria. It would be interesting to investigate, or at the very least discuss, this aspect further.
We thank the suggestion. The ectopic formation of scaffolds in the DNE region along the secondary matrix (2ry) could be one of reasons that progenitors could not reach their destination. Per suggestion by the reviewer, we did quantify the GFAP density at the border of fimbria. Data showed that the GFAP density is not alter in the 2ry, but slightly increased in the fimbria, probably due to the compression by amassed TBR2+ cells at the 2ry (Fig.3o).
We've stated the point in revision. Moreover, bearing the possibility in mind, we did use Nestin-Cre to ablate Kdm2b-CxxC in original submission. As we've tested, Nestin-Cre is mostly not active in cortical hem (CH) derived astrocytic scaffolds at the fimbria ( Supplementary Fig.5b). In the Kdm2b '*/0+-"3%11% hippocampi, significantly more IPCs still accumulated at the DNe and the FDJ, but fewer IPCs at the DG, with the fimbria compressed ( Supplementary   Fig.5c-5g).
5. Regarding the CA phenotype, a reduction of calbindin positive cells is shown in Fig.1. Later, in Fig.6  Regarding these two points, we further analyzed phenotypes in cKO CA and electroporated DG. First, EdU was administered at E14.5 followed by phenotypic analyses at E18.5. In cKO CA1 region, significantly more EdU+ cells resided at VZ and fewer EdU+ cells located at pyramidal cell layer.
Moreover, significantly more EdU-labelled cells expressed TBR2. Second, at E16.5 cKOs, more PAX6+ and TBR2+ cells were detected at the hippocampal neuroepithelia (HNE), where CA neurogenesis occurs. While more PAX6+ cells were proliferative (PAX6+EdU+), a smaller portion of TBR2+ cells were dividing ( Supplementary Fig.7). Thus, loss of KDM2B-CxxC also leads to hampered neurogenesis at the CA region. Third, the effect of Wnt-mix and SFRP2 electroporation on the DG was analyzed in revision. Significantly fewer electroporated GFP+ cells reached the distal (3ry) region of DG, while exon was as efficiently induced. Striking differences in deletion efficiency exist between different alleles, and this is particularly true for inducible Cre which are only active during tamoxifen treatment. Therefore, unless deletion of the targeted allele is shown, these experiments are suggestive but not conclusive.
We agree with the reviewer that "deletion of the Kdm2b exon might not be efficiently induced" because we did not provide direct evidence of exon deletion in the adult DG. We'd like to point out that we've examined This is a very interesting point. First, The RNA-seq analysis indicated that many Wnt signaling genes, including those encoding Wnt ligands and SFRP2, were significantly upregulated in Kdm2b &,1#"2%11% cKO hippocampi, which were validated by qRT-PCR ( Fig.6d-6g, Supplementary Fig.10a-10f).
Because it is known that KDM2B mediates gene silencing by targeting the variant polycomb repressive complex 1 (PRC1) to non-methylated CpG islands (CGIs), we then paid special attention to the change of H2AK119Ub (deposited by PRC1) and H3K27me3 on these de-repressed genes. Data showed CGIs of many de-repressed genes indeed lost the enrichment of 11. Although not pivotal for the main scope of the paper, data shown in Fig.   S9.A-G is not particularly straightforward. The line of the side of panel A ("High in Ctx") appears too short and should go up to Wnt6. The interpretation provided, regarding the differential effect of Wnt signalling dysregulation by KDM2B in the cortex vs. the hippocampus, is not supported by the data presented. Many Wnt genes are indeed clearly upregulated in the cortex. Are there statistically more Wnt pathway DEGs in DG compared to the cortex?
We thank the reviewer for pointing this out. The line marking "high in Ctx" has been moved up to Wnt6 in revised Supplementary Fig.11a. Regarding whether there are statistically more Wnt pathway DEGs in DG than those in the cortex, we did analyze expression levels of all Wnt ligand and receptor genes in hippocampi (HP) and cortex (Ctx). Data showed these genes expressed at a higher level in HP than in Ctx in both control and cKO brains.

Moreover, deletion of KDM2B-CxxC elevated their expressions in both HP
and Ctx (Response fig.4) We thank these suggestions and have mentioned Ring1B and its function in the introduction. In addition, quantification of immunoblotting of histone modifications were done in revision ( Supplementary Fig.11i).
13. Finally, given the unexpected results from these Rnf2 mutants, the potential role of KDM2B for DG development should be better discussed. The graphical abstract suggests a potential role for PRC1 while the Rnf2 mutant phenotype may suggest otherwise? This is an interesting and important point. As the enzymatic component of PRC1, Ring1B is likely responsible for genome-wide deposition of H2AK119Ub. In contrast, KDM2B could selectively control expressions of Wnt pathway genes by targeting variant PRC1 to their CGIs. We've discussed the point in initial submission: "KDM2B likely controls fate determination of hippocampal progenitors by selectively repressing a series of progenitor genes including those in the Wnt pathway via PRC1.1". We further added: "expression alterations of other targets of Ring1B and PRC1 might counter some effects caused by loss of KDM2B-CxxC" and "How KDM2B selectively targets these genes during hippocampal morphogenesis remains to be investigated" in revision.

Minor comments:
We apologize for all these imprecise expressions and have fixed them in revision accordingly with only one exception.
-Line 150: the authors indicate data not shown, but it seems to refer to -In line 280, the length of the EdU pulse for the analysis at E16.5 should be mentioned.
-In Fig. S6 the title should be modified as "abnormal/precocious neuronal differentiation is not responsible…. " -"a tad", "climax", "block of their attenuation" should be replaced.
-Please define all abbreviations The abbreviations list is summarized in Supplementary Table 1.

Reviewer #2 (Remarks to the Author):
In this manuscript, Zhang, et al have discovered that KDM2B plays a crucial role in regulating hippocampal morphogenesis by silencing Wnt signaling in neural progenitors. When the chromatin-association capability of KDM2B was removed in developing dorsal telencephalon, the hippocampus became drastically smaller with disorganized cellular components and structure.
KDM2B mutations in mice resulted in defects in spatial memory, motor learning, and fear conditioning. The migration and differentiation of neural progenitor cells were greatly impeded in the developing hippocampus. The study suggests that KDM2B is essential for proper hippocampal formation and function. The manuscript presents several concrete advances in understanding the role of KDM2B in hippocampal morphogenesis. Overall, I think this is valuable manuscript.
Howerver, there are a few points that could be considered as potential limitations: 1. The behavioral tests used in the study, such as the Morris water maze and fear conditioning, provide valuable insights into the functional consequences of KDM2B mutations. However, these tests have their own limitations and may not fully capture the complexity of behaviors or cognitive functions that could be affected by KDM2B mutations.
We thank the reviewer for raising the important issue. We also realized that although these behavioral tests were commonly used in studies related to hippocampal functions, they have their own limitations and may not fully capture the complexity of behaviors and/or cognitive functions that could be affected by KDM2B mutations. Moreover, as we mentioned in response to Reviewer 1, most Kdm2b-CxxC ckO mice were not willing to explore novel environment. Consistently, in novel object recognition and three-chamber social interaction tests, most cKO animals would stay in corners or circle around walls but were not willing to explore. Therefore, we only included data of non-autonomous behavior tests such as the Morris water maze, rotarod, and fear conditioning.
We have discussed these limitations in revision in the last paragraph of Discussion: "More specific behavioral tests, such as novel object recognition and two-choice spatial discrimination test, could better describe how hippocampal functions were affected by KDM2B mutations".
2. The study found that removal of KDM2B's chromatin association capability in adult neural stem cells exerts no effect on adult neurogenesis of the dentate gyrus. This could be seen as a limitation as it suggests that the role of KDM2B in neurogenesis may be limited to the developmental stage.
According to data revealed in the study, we agree with the reviewer that the role of KDM2B is largely in hippocampal morphogenesis at prenatal and postnatal stages but not in adult neurogenesis of the dentate gyrus. We reasoned that KDM2B is dispensable for adult neurogenesis of DG is because KDM2B is expressed at a relatively lower level in adult hippocampus ( Supplementary Fig.1i). We've discussed the point in revision: "Interestingly,  Fig.1h). Nestin-Cre mice used in this study is from the Jackson Laboratories (stock number 003771) and is active in all neural stem cells from E10.5 ( Supplementary Fig.5b). Nestin-Cre-deltaCxxC pups were born at a ratio lower that the Mendelian expectation and could not be obtained at P7, indicating non-neural effects on loss of KDM2B-CxxC (Response Table 1). The phenotype of Nestin-Cre-deltaCxxC at P0 has been described in Supplementary Fig.5c- Fig.1k-1l).

Authors determined that a strong decrease in GFAP+SOX2+ NSCs, HopX+
NSCs, TBR2+ and DCX+ (which should be quantified as done for NeuN+ and GFAP+SOX2+) cells in the dentate gyrus of adult deltaCxxC animals.
However, they conclude that deletion of CxxC ZF of KDM2B had no effect on adult neurogenesis of the DG based on the conditional deletion in adult animals. Authors should improve the discussion of these results.
First, we've performed quantifications on the numbers of these cells (Fig. 1i, Supplementary Fig. 2i-2k). Second, data from Kdm2b '*/0+-"%.*&()$"2%11% cKO mice did show the deletion of KDM2B-CxxC in adult DGs had no effect on the neurogenesis of the SGZ. In other words, the effects of KDM2B on hippocampus are at embryonic and postnatal stage but not at adult stage. The decrease of adult NSCs in the Kdm2b &,1#"3%11% SGZ is largely due to hippocampal hypoplasia. We reasoned that it is because KDM2B is transiently expressed in embryonic and postnatal hippocampus ( Supplementary Fig.1i).
We've discussed the point in revision.
4. The density (per GCL area or volume) of GFAP+SOX2+ NSCs, HopX+ NSCs, TBR2+ and DCX+ cells should be quantified to demonstrate whether the decrease of these cells is due to the shorter DG.
5. Authors conclude that deltaCxxC mice exhibit hippocampal agenesis. I suggest changing "agenesis" since although smaller, the hippocampus is present in cKO mice.
We apologize for the inaccuracy. In revision, "agenesis" is replaced with "hypoplasia" throughout the manuscript.
6. Figure 2F is not mentioned in the Results section or discussed. What can be concluded of this analysis where no differences in the number of platform crossing were observed in cKO mice. The representative swim path of cKO mice during the probe trial ( Fig 2C) should be change; it does not seem representative based on the graph.
We apologize for not mentioning the panel and agree that there was no statistic difference regarding the number of platform crossing in cKO mice. We therefore replaced representative swim path of the cKO mice and mentioned it in revision (Fig. 2c). Nonetheless, cKO mice spent less time in the SE quadrant where the platform located (Fig. 2g), indicating that the cKO mice had impaired spatial memory.
7. Line 235-237: "Together, loss of KDM2B-CxxC impedes migration and differentiation of IPCs, hence proper production and localization of granule neurons during hippocampal formation." How did authors conclude with the results of Figure 3 that there was an impairment in differentiation. The effects in PROX1+ cells could be caused only by the altered migration; and is it possible to exclude a proliferation impairment? Total number of EdU+ cells and the percentage of EdU+ cells positive for PROX1 should be evaluated.
We agree the reviewer that based on data presented in Figure 3, the decrease of PROX1+ DG neurons could be largely explained by impeded migration of TBR2+ progenitors. The sentence was then changed into "loss of KDM2B-CxxC impedes migration of IPCs, subsequently resulting in hampered production and localization of granule neurons during hippocampal formation" in revision. We'd like to point out that the number of PROX1+EdU+ cells was also reduced by 27.1% in cKO hippocampi (Fig. 3d), which suggests impaired neuronal production. We also quantified EdU+ cells and the percentage of EdU+ cells positive for PROX1 in the DG. Data showed 39.5% fewer EdU+ cells were detected in cKO DGs (Fig. 3e), whereas the ratio of EdU+PROX1+/PROX1+ was not significantly altered. We'd like to point out since it's the birthdating (7 days pulse-chase) experiments, we could not infer that in cKO hippocampi, cells born at E15.5 (labelled by EdU) had fewer chances to became granule cells and located at the DG. The issue of proliferation will be addressed in the following section.
What do authors mean by delayed differentiating trajectory? Considering that as shown in Fig 4 "the differentiating rate from RGCs to IPCs (PAX6+TBR2+/PAX6+) at FDJ is 60% higher in cKOs".
9. Line 298-301: "Together, KDM2B regulates hippocampal morphogenesis by controlling multiple behaviors, including coordinated RGC to IPC differentiation, migration, and divisions of neural progenitors (Fig. S6C)." What is the evidence to conclude that KDM2B regulates divisions of neural progenitors? The discussion of the evidence supporting the specific effects (or roles of KDM2B) on migration, differentiation, proliferation should be improved.
(1) These are two related and important comments concerning the division and differentiation of hippocampal progenitors. We apologize for not describing and discussing clearly enough. The neurogenesis of DG neurons follows the "RGC to IPC to neurons" trajectory. At E16.5, most PAX6+ RGCs were localized at the DNe, while TBR2+ IPCs were more evenly distributed along the DNe-FDJ-DG migratory/differentiating path. Data in Fig.4 showed in E16.5 cKO hippocampi, more PAX6+ RGCs reside at the DNe and dividing more, which give rise to more TBR2+ IPCs at the DNe. In contrast, much fewer RGCs and IPCs could be detected at cKO DGs, and these RGCs and IPCs barely divide. At P7, most TBR2+ IPCs reside at the DG and divide, whereas knockout of KDM2B-CxxC results in significantly more IPCs locate at SVZ and FDJ, indicating 'delayed neurogenesis". In summary, the inability of RGCs and IPCs to migrate along the DNe-FDJ-DG path and to generate progeny in the DG at proper time point was summarized as "delayed differentiating trajectory". We've extensively modified related texts in revision.
10. Please indicate in the results section what Wnt ligands were used in the "Wnt-mix".
We've indicated Wnt ligands used in the Wnt-mix in revision.
-In the description of the Morris water maze results, it should be indicated that the probe trial was carried out on day 6.
-There is no sequential order in the description of some Figures - Figure S3 legend, line 75 "(D)" not "(A)".
-Fig3I, y-axis "Total" not "Totol" -A brief description of the markers used should be given in the results section when describing the IFs (e.g. ZBTB20).
We apologize for these errors and have fixed them in revision accordingly.
Brief descriptions of the markers used have been added in revision.
In this revised version of the manuscript, the authors have performed a substantial number of experiments in response to our comments. In particular, more quantifications have been added  to better describe the phenotype of mutants. Importantly, more in utero electroporations have been performed (Fig.7), strengthening the role of increased/ectopic Wnt signalling in abnormal migration and neurogenesis of DNE progenitors. We think these data have improved the manuscript and that that all our concerns have been addressed. A minor issue that could be improved in Sup. Fig.1j would be to show split channels on the immunofluorescence so that so that Kdm2b co-localisation with TBR2 is better seen.
Reviewer #2 (Remarks to the Author): Overall, I feel the authors have addressed our concerns" and no further questions.
Reviewer #3 (Remarks to the Author): The authors have addressed all my comments and concerns.